I- . FIGUW 4 GAS CIIROMATO(;fiAPiI OF URINARY ACID PROPILE A. PATIKIT ACIDOTIC (Z-17-73) I\" !`A'I'T)`yr' vfq."; 1, ( 2,. 1 (l-71 1 l.fIf! n.52 3.51 f-l.23 Q.4P -!I.51 ').7c, 2.7') n.74 I , :: 1 3 "I, Areas A r-as Arr-as Areas Arwis hrfvs Areas Arcas At-pas Art-as Areas 11349.2/ 21382.5 l&LX/ 109h7.3 6272.4/ 6054.7 39;. 21 I;!i53.7 R35,1/ 67621.3 7cn3.1/-330es.r 116?.7/ 2712.q 3293.4/ 31f?4.7 2920.1/ 5971.8 22El.E/ 5751.0 lcZE3.1;/ 19353.8 PATIC! = 0.5308 Locattan Error - 9 ?????? ?? o 0.1771 Locatifh rrrar =`-2 F4T PAT PAT PAT I 1.0277 0.107G 0.1235 -0.2238 0.4270 FIGURE 5 ANALYSIS OF 12 AMINO ACIDS IN URINE USING MASS FRAGMXNTOGRAPHY Location Erroi = 2 Lccntfnn Error - -1 loc?tinn Frrnr = 2 Locatfon Crror = 2 LocatIon Error = 0 Location Error - 0 Lor?&an 'rror = -2 Locattnn rrror = 0 Loci+tlan Frror - -1 THE SIMULTANEOUS QUANTITATIPN OF TEN AMINO ACIDS IN SOIL EXTRACTS BY MA& FRAGMENTOGRAPHY W. E. Pereira, Y. Hoyano, W. E. Reynolds, R. E. Summons and A. M. Duffield Department of Genetics, Stanford University Medical Center Stanford, California Running Title: Mass Fragmentography of Amino Acids Address for Proofs: Dr. A. M. Duffield Department of Genetics Stanford University School of Medicine Stanford, California 94305 Received P- 5-z The analysis of amino acids from terrestrial and extraterrestrial sources is becoming increasingly, important (l-5) , The need for a specific, I sensitive and rapid method of quantitation is desirable. The methods currentiy employed for amino acid analysis involve ion exchange procedures (6,7) or gas chromatography (8-10). These techniques, although of immense value, are limited by their non-specificity for the absolute identification of any substance responsible for a gas chromatographic peak. In the present communication we report an absolute, unambiguous method for the positive identification rnd quantitation of ten amino acids present in soil extracts using GLC-mass fragmentography. In mass fragmentography the mass spectrometer is used only to detect certain preselected ions known to be characteristic for each compound being quantitated, and the internal standard. The technique of mass fragmentography . . using sector mass spectrometers is usually restricted to the simultaneous monitoring of up to three integer mass values (11, 12), although with one instrument five ions were used spectrometer up to eight ions have analog signals monitored (14). We (13) o Using a.quadrupole mass been selected and their respective now wish to report the modification of the gas previously conqo~ of chromatography-quadrupole mass spectrometer-computer system described (15) for the simultaneous monitoring under computer ., . . . , the ion currents from 2.5 cc\nje br*w-- WIICS C Ld 750 i- con+tst 2 r;sc.l;Eetdcnteger mass _ values. -pjcsc via us cc- ;yL UVIIlCbIL 4or n`$l &8y--io+j ;I:;*(j SC' P*r.tr'XIf required this number could be increased by suitable alteration of the computer control programs. Specifically we wish to report the application . of thii system to the quanti'tation of ten of the amino acids present in soil . ___. --. _- ., extracts . Reagents: A deuterated amino acid mixture was supplied by Merck Laboratory Chemicals (New Jersey). 1.25N HCl in n-butanol, 25% (v/v) trifluoroacetic anhydride in methylene chloride and Tabsor column packing were obtained from Regis Chemical Co., Illinois. A standard amino acid solution was purchased from Pierce Chemical Co., Illinois. Equipment: A Varian model 1200 gas chromatograph was coupled by an all glass membrane separator (16) to a Finnigan 1015 Quadrupole mass spe-trometer which was interfaced to the ACME computer system of the Stanford University Medical School (15). CLC separations were conducted using a 6 foot by 4 mm. (I.D.) coiled glass column packed with Tabsorb (Regis Chemical CO.). The flow rate of the carrier gas (helium) was 60 ml/minute. . . The uniqueness of the mass spectrometer instrumentation lies in the -_- modified computer software (program) used. The hardware is the system previously described (15) and as:umes an operating cycle of: (a) transmission of a control number, N, from the computer to an interface controller which sets the quadrupole mass analyser to a particular mas-s point in the m/e continuum. -- (b) an integration of the ion.signal for a pre-set period, T (integration time = 8 milliseconds in our work), and (c) computer reading of the integration value with a twelve bit A + D conversion. . * ' For the recording of normal mass spectra N is selected such that successive cycles result in m/e values of 1,2, . . ...750. At the beginning of each -- day the instrument is calibrated using a reference compound. Idiosyncracies of the IBM 360,/50 to IBM 1800 computer data paths dictate that the mass s values be buffered into groups of 250.% For normal g.c.-m.s. procedures the operator is allowed to select a mass range of 1 to n x 250 (n = 1,2, or 3 buffers). For mass fragmento- graphy n is set to zero and instead,a "precision collect" buffer of 250 control-data acquisition cycles is employed. The operator must then enter the pre-selected m/e values he wishes to scan. When the precision collect -- buffer is constructed, 10 cycles are allocated to each m/e value selected. -- The first of the 10 cycles sets N to K -4. In The returned integrated ion measurement is discarded; this cycle serves only to slew the quadrupole electronics from anywhere in the m/e continuum to the mass region of -- InterFst. The additional 9.cycles are used with N = N,-4....Nm.....Nm+4. The returned values represent a set of readings about the m/e value of -- Interest + 0.5 amu. The center three points are then smoothed with a five point qudratic function (17). The highest value of these three smoothed points is then selected,as the precision collect value. Thus , small drifts in calibration are corrected and a signal average obtained. Finally ,i the abbreviated "spectrum" of 25 precision intensities for each m/e are filed on disc. a- Such a "spectrum" is recorded every 2 seconds and a summation of all the ion intensities is used to cou$truct the ion chromatogram shown in Fig. 2. Inditidual ion chromatograms can also be constructed if required (Fig. 3). A threshhold is established from the ion currents before and aftet each gas chromatographic peak and a computer program performs integration of the ion currents under each peak. Procedure 1 g of sieved, air-dried soil (Stanford University garden soil) was refluxed with 6N HCl (10 ml) for 20 hrs. The mixture was filtered and the residue washed kth 1N HCl (5 ml). The combined filtrate and washings were extracted with chloroform (4 x 10 ml) and the aqueous phase evaporated to dryness. The residue is dissolved in water (5 ml) and passed through a column of "Ion Retardation Resin" AG 11-A8 (So-100 mesh, 1 x 21 cm). The amino acids were eluted with water (50 ml) and the eluate evaporated in vacua to dryness. The residue is dissolved in water (5 ml) and placed on a column of p-57 cation exchange resin (AC SOW-X12, SO-100 mesh, 1 x 21 cm) and washed with water (50 ml) to remove neutral and anion contaminants. The amino acids were eluted with.4N NH4?H (80 ml) and the eluate evaporated to dryness. The residue was disiolved in water and made up to a volume of 4 ml. A portion of this solution (1 ml) was used for the amino acid analysis using an amino acid ,analyser. To another 2 ml of the processed solution was added 2 ml of the deuterated amino acid standard solution (100 mg in 100 ml of O.lN HCl) and the mixture evaporated to dryness. The residue was refluxed with 1.2 N HCl in n-butanol (1 ml) for 30 min. and evaporated to dryness in vacua. To the residue trifluoroacetic anhydride in methylene chloride (0.7: ml) was added and refluxed for 10 min. The solution was evaporated to dryness at room temperature and the residue dissolved in ethyl acetate (100 ~1). An aliquot (1 ~1) was injected into the injector port of the gas chromatograph and the oven kept at 100" for 1 min. when it was programmed at 4"/min. to 220'. To each of 4 tubes containing 2 ml of the deuterated amino acid standard solution (100 mg in 100 ml of O.lN HCl) was added 150, 200, 250 and 300 ~1 respectively of a standard amino acid solution (2.5 ,,moles -e of each amino acid per ml). The solutions were mixed and evaporated 'to dryness. Each residue was derivatized by the above method and an aliquot of each (1 l.J) injected into the gas chromatograph which was operated under the conditions described above. This procedure . . . . was used to construct a standard curve for the quantitation of each am&o acid. A typical standard curve is shown (Figure 1) for glutamic. acid. p- 5-z RESULTS The N-TFA, 0-n-butyl derivative was chosen for the derivatization s of amino acids for two reasons.. Firstly, these derivatives have 14 excellent glc separation characteristics @$j and secondly the selected . characteristic fragment ions of the deuterated and non-deuterated derivatives do not interfere with each other, nor with other a-amino acids. Table I records the individual ions monitored for quantitation in the mass spectra of each of the deuterated and non-deuterated amino acids. Thezomput~tegrates-the-intensity of the deuterated and Be-ratio-of-their-respective-peak-areas7 Our results of a typical soil analysis are compared with those from an amino acid analyser in Table II. The higher value obtained with lysine by the amino acid analyser is due to a ninhydrin positive substance in soil interfering with the quantitation of lysine. In this respect mass fragmentography is superior to the amino acid analyser in that using a mass spectrometer as detector only characteristic pre-selected ions are detected and quantitated and any impurity present' under the same gas chromatographic peak is not measured* A summation of 20 such characteristic ions was plotted as an ion chromatogram of a derivatized soil sample and is shown in Fig. 2. Preliminary experiments showed that when the deuterated amino acid mixture was added directly to the soil sample extensive hydrogen- deuterik exchange occurred during acid hydrolysis of the soil extract. The removal of the isotopic label was catalysed by the hot mineral acid in presence of excess mineral used in the soil hydrolysis step. Fox and collaborators have reported (4) a similar finding concerning the _ - decomposition of amino acids in soil upon direct acid hydrolysis. In the present work the deuterated amino acid mixture was added just before derivatization (i.e. after hydrolytic extraction of the soil) in order to avoid this problem. However, in cases where it is necessary to quantitate the free amino acid content of complex mixtures, such as In serum or urine samples, the deuterated amino acid mixture may be added sample before processing without any deleterious Although only ten amino acids present in soil were quantitated the method can be extended to all the normal amino acids found in protein. The deuterated analogs of arginine, histidine, serine, threonine and tyrosine are commercially available. Appropriate deuterated analogs of methionine, tryptophane, cysteine and cystine would have to be chemically synthesized from the appropriate precursors. In these instances at least two deuterium atoms should be incorporated in non-exchangeable positions so that for the characteristic ion chosen the P + 2 peak is separate from the 13 C isotope contribution of the unlabeled amino acid. Furthermore, the deuterium substitution need not be quantitative (>90%) provided the same characteristic ion of that deuterated analog is used for the construction of a standard curve such as Figure 1. -Lx Instrument analysis time is approximately one hour and with our system we have been able to achieve accurate quantitation with samples containing as little as 10 nano'grams of an amino acid, SUMMARY A specific and sensitive method for the identification and simultaneous quantitation by mass fragmentography of ten of the amino acids present in soil has been developed. The technique uses a computer driven quadrupole mass spectrometer and a commercial preparation of deuterated amino acids is used as internal standards for purposes of quantitation. The results obtained are comparable with those from an amino acid analyser. In the quadrupole mass spectrometer-computer system used up to 25 pre-selected ions may be monitored sequentially. This allows a maximum of 12 different amino acids (one specific ion in each of the undeuterated and deuterated amino acid spectra) to be quantitated. The method is relatively rapid (analysis time of approximately one hour) and is capable of the quantitation of nanogram quantities of amino acids. . . . . . ACXNOWLEDGMEXTS This research was funded by the Planetology Program Office, Office of Space Science, NASA Headquarters under grant NGR-05-020-004. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. '13. 14. POCKLINGTON, R., Anal. Biochem. 45, 403 (1972). TAYLOR, R. and DAVIES, M. G., Anal. Biochem. 51, 180 (1973). - HALPERN, B., WESTLEY, J. W., LEVINTHAL, E. C. and LEDERBERG, J., Life Sciences and Space Research - North Holland, Amsterdam, 239 (1967). HARE, P. E., HARADA, K. and FOX, S. W., Proc. Apollo 11 Lunar Science Conf. 2, 1799 (1970). = KVENVOLDEN, K., LAWLESS, J. G., PERING, K., PETERSON, E., FLORES, J., PONNAMPERUMA, C., KAPLAN, I. R. and MOORE, C., Nature, 228, 923 (1970). Z SPACKMAN, D. H., STEIN, W. H. and MOORE, S., Anal. Chem. 3&, 1190 (1958). HAMiLTON, P. B., Anal. Chem. 35, 2055 (1963). GEHRKE, C. W., KUO, K. and ZUMWALT, R. W., J. Chromatog. 57, = 193 (1971). GEHRKE, C. W., ZUMWALT, R. W. and WALL, L. L., J. Chromatog. 37, 398 (1968). . . . ._ JijNSSON, J., EYEM, J. and SJGQUIST, J., Anal. Biochem. 51, 204 (1973). - HAMMA!, C. G., HOLMSTEDT, B. and RYHAGE, R., Anal. Biochem. 2, 532 (1968). _- HAMMAR, C. G. and HESSLING, R., Anal. Chem. 43, 298 (1971). -- - SNEDDEN, W., PARKER, R. B. and WATTS, R. E., Int. Conf. on Mass -- Spectrometry Brussels, 31&. i 4 Sept. 1970, Vol. 5, Institute - of Petroleum, London, and Elsevier, Amsterdam, 1971. p. 742. . . KNIGHT, J. B., Finnigin Spectra, &, No. 1 (1971). .lS.. REYNOLDS, W.: E.; BACON;& A;, BRIDGES, J.. C., COBURN;T. C.,. HALPEPN, B., . LEDERBERG, J., LEVINTHAL, E. C., STEED, E. and TUCKER, R. B., Anal. .`, . Chem. 42, 1122 (1970). = 16. HAWES, J. E., MALLABY, R. and WILLIAMS, V. P., 2. Chromatog. Sci. 2, 690 (1969). (%H. GEHRKE, C. W. and STALLING, i. L., Separation g. 2, 101 (1967). 1q.6 PEREIRA, W. E., BACON, V. A., HOYANO, Y., SUMMONS, R. and DUFFIELD, A. k., Clin. Biochem. (In Press). Table I. CHARACTERISTIC FRAGMENT IONS SELECTED FOR MASS FRAGMENTOGRAPHY OF UNDEUTERATED AND-DEUTERATED . N-TFA-0-IJ-BUTYL-AMINOrACIDS. Amino Acids i Fragment Ion VAL GLY ILEU LEU PRO PHE ASP GLU LYS CH3CH=kICOCF3 (m/e 140) CD3CD(NH2)COOH I-C3H7CH=zHCOCF3 (m/e 168) I-C3D7CD(NH2)COOH CH2&COCF3 (m/e 126) NH2CD2COOH C2H5CH(CH3)CH=zHCOCF3 (m/e 182) C2D5CD(CD3)CD(NH2)COOH i-C3H7~2CH=kXOCF3 (m/e 182) I-C3D7CD2CD(NU2)COOH kCOCF3 (m/e 166) C6H5CH=CHCODHl + (m/e 148) BuOOCCH2CH&XOCF3 (m/.e 240) HOOCCH2CH2CH=kKOCF3 (m/e 198) CX2-CHCH2CH2CH-&COCF3 (m/e 180) Deuterated Amino Acids D C6D5CD2CD(NH2)COOH HOOCCD2CD(NH2)COOH HOOCCD2CD2CD(NH2)COOH Fragment Ion CD3CD&COCF3 (m/e. 144) . . I-C3D7CD=hCOCF3 (m/e 176) CD2=iHCOCF3 (m/e li8) C2D5CD(CD3)CD=zHCOCf+3 x+/e 1%) i-C3D7CD2CD=hCOCF~~~(m/e 192) D D K2 hi -COG3 (m/e 173) DD ; D C6D5CD=CDC001q+ (m/0.155) , BuOOCCD2CD=hCOCF3 (m/e 243) HOOCCD2CD2CD=$HCOCFj, (m/e 203) CD2-CDCD2CD2CD&O~~3 (m/e 188) LEGENDS TO FIGURES Fig. 1. Standard curve for the quantitation of Glutamic acid. Fig. 2: Typical ion chromatogram of soil amino acids. Ftg.3. fv\',sr Fc=p e-2 Q+ub& -i--*u- O- f . Table II. ANALYSIS OF AMINO ACIDS IN SOIL (pg/g SOIL) Mass Fragmentography Amino Acid Ala Val GUY Ileu LeU Pro Phe Amino Acid Analysis 206.5 148.3 215.4 95.4 154.2 143.4 80.3 218.3 227.0 129.7 Bl 198.7 151.0 ASP Glu LYS S96.8 100.4 152.1 141.4 80.5 217.1 217.2 115.3 #2 202.7 150.5 201.6 100.2 149.7 142.8 80.7 219.8 215.6 113.5 83 198.3 149.9 201.3 92.3 154.2 141.2 80.0 219.9 214.1 114.9 P-Lb Fig. 1. GLU - 4dded D5^GLU - 3 J 3 E i, I IIC. 3 A Lomputer Uperated Mass Spectrometer System W. E. Reynolds, V. A. Bacon, J. C. Bridges, T. C. Cohurn, Berthold Halpern, Joshua Lederbcrg, E. C. Levinthal, Ernest Steed, and R. B. Tucker Department o$ Genetics, Stan$ord University School of Medicine, Stanford, Calif. 94305 An integer resolution mass spectrometer-computer system has been developed in which the computer controls the "scan" of a mass spectrometer. In this system, the computer queries the user for operating parameters which are then translated into control functions which operate the mass analyzer. The spectral information acquired from the mass spec- trometer is made available to the chemist within min- utes in an on-line graphic system. processing of GLC effluent are given. Examples of the THE USE OF htAss SPECTROMETRY has been hampered by the lagging development of a fast and convenient method of re- ducing the spectral output of the mass spectrometer (MS) to numerical data. Usually the operator must convert a MS chart recording, which is an analog plot of intensity cs. time, to a digitized plot of intensity cs. mass number. Because of instrument instabilities, wide range of signal amplitudes, large amounts of data, and other operational ditficulties, (I; 2), it is often dificult and time-consuming to establish all the correct mass peak identifications. One aid is to use a reference compound (3) tither prior to the run or as an in- ternal standard with the unknown sample. By counting from known mass peaks, unknown spectral peaks can bc idcntificd. However, the processing of data by this tczhnique is still a formidable task and it may take several days to accumulate all the information from a gas chromatograph-mass spec- trometer (GLC-hIS) run. Several workers have demonstrated MS-computer systems in which the computer monitors and records digital data from a MS. In most of these applications the mass spcc- trometer has operated indepcndcntly oi the computer, scannmg in some time dcpcndcnt mode. mcnsuring ion intensities at all points within the range of (500 to 5000 samples per second) and afterward performs the computations required to reduce the large amounts of digital data to useful information (4-7). Much instrument time and sampling effort is ex- pended in the intervals between integer peak positions where there is little or no information. One system that improved (1) K. Biemann, "Mass Spectrometry." McGraw-Hill, New York, 1962, p 10. (2) J. Lederberg, E. Levinthal, and Staff. "Cytochemical Studies of Planetary Microorganisms Explorarlons in Exobiology," IRL Report No. 105-1, Instrumenration Research Laboratory, Department of Genetics, Stanford University School of Medi- tine, April 1966 to October 1966. NASA Accession No. N66- 34195. - (3) J. H. Beynon. "Mass Spcctrometry and Its Applications to Organic Chemistry," Elsevicr Publishers. Amsterdam, 1960, p 44. (4) R. A. Hitcs an K. Biemann. ANAL. CHEhq., 39,965 (1967). (5) ILGd., 40, 1217 (196s). (6) R. A. Hitcs, S. Markey, R. C. Murphy, and K. Biemnnn, 16th Ann. CO/I./. ,lfrw Spcc/rarwrr.v Aliid To,Cs, ASTM E-I J, Pittsburgh, Pa., hla>, 196s. (7) R. B. Tucker, "A hlass Spcctromater Data Acquisition and Analysis System." IRL Report No. IOG?, Instrumcntntion Research Laboratory. Depnrtmcnt of Gcncticb. St:tniorcl Um- versity School of Medicine, NASA Acc:ss~on No. NOS-25743, 1968. upon this latter ineficiency used step switches to step the scan from position to position (8). We now describe a MS-computer system, suitable for routine laboratory use, in which the computer controls the operation of a quadrupole mass spectrometer (9, 10). In this system the "scan" is calibrated by relating known mass positions of a reference compound to a computer g~ncxtcd control voltage (V,). R, is gencratcd as the result of a number N, serit from the computer to a Digital-to-Analog (D-to-A) converter in a MS-computer interface. The parameters of this V,, or the N for each integer mass position, arc de- termined by a computer program and stored in memory. The subsequent use of this information allows the computcr- directed MS output to be recorded directly as mass,chnrgz (nz/r) cs. intensity. On request, this data is then made al,ail- able to the operator in an on-line system. The use of this computer-.MS interaction, combined with the decision-making ability of the opcra+?r, permits n sig- nificant saving in data processing costs. Furthermore, a much larger duty cycle of analyzer "on peak" time is obtain- able, resulting in the detection of more ions for a given rnxs position than is possible in conventional time based scanning. The new MS-computer system has at least thrcs unique features. There is a hardware control intcrfncc to connt':t the MS intimately with the computer; there is an improv:d etlicicncy of information acquisition from spectral psahs that are limited in ion production rates; and there is a uscr- oriented control and data presentation system that csnc.xls the foregoing details from the operator, but prssen:s 1i7c gxr with prompt and concise data which include normalirsd mass spectral plots. The described system has evolved through three mass spectrometers, three computers, and two basic computer pro- grams (I I, 12). The later systems have greater range. scnsi- tivity, and convenience, but they all have a common concept. Therefore the description that follows will be concrprunl rather than specific to any one configuration. (8) H. L. Friedman, H. W. Goldstein. and G. A. Griffith. "\lnjs Spectrometric Thermal Annllsis of Polymrr DCCOnlpOhiIiJn Products," 15th d~vr. CU/I~: .\fcrss S~cctronre!r.v rlllrc*ti 7'0,~jcs. ASTM E-Id, Denver, Colo., hfay 1967. (9) W. E. Reynolds, "A Small Computer Approach to Lou Resolution hlnss Spectrometry." Pacitic Conference on Chc:l:ls:r> and Spectroscopy, Anaheim. Cnlif.. November 1967. (IO) W. E. Reynolds. T. B. Coburn. J. Bridges. and R. Tuc`ker. "A Computer Operated ,Ilass Spectrometer S~stcm." IRL Report No. 1062. lnstrumcnration Research Laboratorv [)c- partment of Genetics, Stanford Univcwty School of \lc::c!zc. NASA Accession No. N68-l ! S69. Nov. 1967. (11) W. E. Reynolds. R. B. Tucker, R. A. Stillman. and J. C. Bridges, "hlass Spcctromcrers In a Time Shxcd C:xn:~u!~r Environment," I7 t h rlrur. Co,rjI iIl:,ss Sprcrror,~c*tr~ .~li!;,x Topics, ASThl E-14. 1969. (I?) J. Lectcrberg. E. Lcventh:ll. and StatT. "C! tochmxnl Stt~d~c; ot Planetary hlicroorgcanisms Esplorshons in t\;olxoioc~ ." II;L Report No. 1076. Instrumcntntion Rcscarch Labornror). I):- partmcnt of Gcnctizs. Stnnf'urd C'mvcrsity School of X1:, I;,!::. October 1967 to r\prll IYM. Appcndiu A IS a ~LI!~~I~IICLI IC;':.IIL of the abovc "hlass Spcctrometcrs in 2 Tuw Shurcd t. II\ iron- menl," NASA Accession No. N6S-ZYS-IG. Reprinted from ANALYTICAL CHEMISTRY, Vol. 42. Papc 1112, Scptentbcr 1970 Copyright 1970 by the Amcricm Chemical Society and rcprintcd by permission of the copyright owner P-7 1 , The present system is operating with a Finnigan 1015 quadrupole MS and a Varian Aerograph GOOD chromato- graph. The same computer programs and a similar interface were also operated successfully with a Bendix Time-of-Flight (T-o-F) MS (13) and an EAI qundrupole (14) MS. In all cases the GLC-MS, the teletypew/riter, and the digital plotter were situated in a wet chemical laboratory. A schematic diagram of the GLC-MS combination is shown in Figure 1. The emuent from the gas chromato- graph, equipped with a flame ionization detector, first passes through a variable splitter that diverts between Ii3 and *I? of the flow through a Biemnnn separator (15) and into the MS. A solenoid-actuated valve in this line helps to keep the large initial solvent peak from entering the MS system. A reference gas reservoir containing a fluorine compound at a vapor pressure of approximately 3 X IO--' Torr is also in- corporated in the system and is connected to the MS by another solenoid valve. The computer, tin the interfacing electronics, has direct control of gas valves, and can valve in or shut off the reference gas whenever it is needed for the calibration routine. These valves were constructed in our shops in such a way that the back side is open to the vacuum system when the valve is closed. This avoids the common pressure burst when conventional valves are opened to a vacuum. The right side of Figure 1 illustrates the major components and functions of the interface. This computer-MS interface was built in our Instrumentation Research Laboratory (16) and contains all the electronics not normally supplied with a standard configuration IMS or computer. Al1 of thcl operating parameters of the hlS are, or may be. controlled by a digital word (binary number) sent from the computer. The principal control is ricl fhe "N" register to the D-to-A converter. The analog signal, V,, from the D-to-A sets and holds the mass analyzer to pass ions of a predetermined ))I e. Alternately the digital output may be coded to operate auxiliary control functions, such as actuate valves. set amplifier gains, the low speed multiplexer, or enable the digital plotter. STlEV'3 ONll33NN03 UOHS 1 The characteristic method of controlling the nl:e passband and taking measurements while the mass analyzer dwells upon a m/e value converts what is normally measured as a time dependent parameter, to a stationary signal. This statistically stationary property of the signal enables the em- ployment of full integration to enhance signal to noise. Both the electrometer and the integrator are standard commercial FET operational amplifiers of the 550.00 class. The time allowed for integration and the operation of the integrator reset are controlled by signals (numbers) from the computer to the "T" register. The output of the integrator is sampled, held, and read ciu the Analog-to-Digital (A-to-D) converter. Auxiliary signal sensing is provided by the low speed multiplexer. This is useful to determine the automatic settings for self calibration,or may be used to record tempcra- ture, pressure, etc. These sense functions, plus some valve (13) D. B. Harrington and R. S. Gohlkc. "High Resolution Time of Flight Mass Spectrometers." 10th .Irw. Coqj: ,Iltrss Sp~wrum- erry A//id Topics, ASTM E-14. New Orlcnns. La.. 1962. (14) W. I'& H. I'. Keinhard, and U. van Znhn, Z. Plrys., 152, 143 (1958). Figure 1. The GLC-AIS instrumentation and the electronics interface to enable computer systems integration control and checkout functions, are controlled by the "C' register. (IS) J. T. Watson and K. Bicmnnn. ANAL. CIIEM., 37, WI (1965). (16) W. E. Kqnolds, J. C. Hric!gcs. R. B. Tucker, and T. U. Co- burn, "Computer Control of Xfass AnalyLcrs," 16th .&III. Co\!/: Mass Sprclrowe~ry d//M Topics. ASTXl E-14, Pittsburgh. Pa.. 1968. Thcrr are no manual opcrntor control functions in :lny o' the above steps. The control is nccompli>hcd iIt tl~ I 11 writer keyboard. This keeps the system llcsiblc ant! IILI,L,. it indcpendcnt of the idiosyncrasies of indi\,idual con~put~ I P-1' ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 . 1123 COMPUTER DIALOGUE (The rcser's respome is underlined) OPTION = collect EXPERIMENT; = test - T=g MODE = single PLOT, TYPE, or FILE = plot - BY MASS, AMP. or ENTIRE = entire FROM MASS = 1 g,;g n PLOT, TYPE, or FILE = 1 T=I EXPExIMENT,+' = MS26x15 --~ CONTINUATION = yes T=6 - MODE = continuous # OF SPECTRA = 130 FILED IN POSITION 930 to 1070 T=/ EXPE`i%IMENT; = / OPTION = sum - EXPn = MS26x15 _~--- FROM SPECTRU,M = 9-U) TO = 1020 - FROMXSS = ilo TO MASS = 500 PLOT = yes - EXP,+ =, 1 OPTION = plot EXPERIMEtii+ = XlS26x15 _- - FRO&M SPECTRU.M = 957 TO = 957 - &OM%iASS = 1 TO MASS = 340 - - interrupt lines and/or individual computer characteristics. This straightforward system definition makes the software design much like conventional computer programming rather than encouraging intricate techniques highly dependent upon the specific hardware. Thus the system is not oriented specifically to any given computer. It has operated on an early model LINC (17) com- puter with 2K words of 12 bits. memory, and on a time-shared, locally programmed, IBM 360;50, butTered with an IBhl 1800 (18). In all cases the computer was somewhat remote, separated by some 500 ft of cable from the rest of the in- strumentation. The system is very economical of computer resources. Most of todak's small general purpose computers would be able to operate the described functions if it were desired to avoid time-shared computer dependency. Some (17) R. W. Stacy and B. Waxman. "Computers in Biomedical Re- search." Vol. II. Academic Press. New York. N. Y.. 1965. DD 35-66.. . . (18) W. J. Sanders, G. Breitbard. G. Wledcrhold. er (I/.. "An Ad- vanced Computer for hlcdicnl Research." Full Joiut Cortrpwr Colt/. Proc., ACM, Anaheim, Calif., 1967, p 497. COhlMEm The user reauests the data collection chase. A catch-all name, "test". is given; the spectrum will be used simply for a systems check. An integration time of 35 milliseconds per peak is requested. Only one spectrum will be taken (single mode). The data is acquired after this answer. The user can plot, type. or file the data col- lected; here a plot is requested. The user can plot selected masses, the highest intensities, or the entire spectrum within requested limits. The user indicates the limits. A QUICK plot omits annotation, etc. Note that "y" and "n" mean "yes" and "no". The user completed the checkout and now wishes to proceed with the experiment. The "/" is used to backup through the conversation. An existing experiment name is given here. The user confirms that the spectra are to be added to the eslsting experiment file. The user requests that spectra be collected continuously until 130 are taken. Data collection is complete. The user then wishes to sum the elements of each spectrum to produce a "total ion" plot, analogous to a GLC trace. Mass position 40 to 500 are summed for each of the collected spectra. The "total ion" curve is now plotted. (Figures 6 and 7 are illustrative of this &tiple dialogue.) Guided by the total ion plot. the user will plot interesting m3ss spectra. Only the spectrum filed in position 957 is chosen. The plot (Figure 17) is drawn and normalized to the base peak, nlje = 31. Figure 2. An example of the user-computer dialogue during operation sort of magnetic storage for object code programs and data storage is most desirable. DEC-t)-pe tapes ha;-c be:n uzd on the LINC system and disc packs on the IB\l s\-stem. THE SOFIV.IRE STRUCTURE The objectives of the software are to operate and conrrol the MS, acquire data from the MS, process and prcsznr rhis data in a manner useful to the chemist, and provide ctrtn:n control and information to aid in maintaining and ser\-l:ing the instrument. With the program loaded into the computer, the Csfr requests any one of several functions (see Table I) by t!Fing the name of that function. The computer responds \x::h a series of prompts (see Figure 2) to elicit user m;lcrocomm:!ntis. The computer then generates the detailed conrrol funz!ons to perform the assigned task. At the comp:.!- tion technique makes the system both tlesiblrt an;l rl) self-instructing. 1124 . ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 P- 72 Table I. A List of Program Options (1) CALIBRATE: Creates an accurate N Table. The .V "urn- hers which correspond to the peaks in the reference gas are used as the end points of a piecewise linear interpolation pro- cedure for calculating a complete .V Table. (2) COLLECT: Is the primary data collection step. It is here that the 750 N Table values are sent to the MS and the 750 m/e intensities rccordcd. This operation can be repeated at five-second intervals as the data are tiled on disk under an e.y- periment name. (3) TYPE: Allows the user to print out spectral data by indicating what spectra in a given file are to be reviewed. The user can request that the amplitudes of particular rtr/e positions be typed ; that a given number of the highest amplitudes be typed, or that a consecutive number ot' them over a given range be typed. (4) PLOT: Enables the user to have bar graphs produced by the computer controlled digitai plotter. The amplitudes to be plotted can be selected with the same flexibility as described in TYPE. (5) SUM: Produces a plot of the total ion current over a series of gathered spectra. All responses of a spectrum are summed to produce one datum point on the plot. This plot corre- spondsclosely with the GLC output when runrung with theGLC. (6) TRACE: Produces a record of a spectrum similar to the normal chart recording output. The analyzer is sampled at all N values (about 10 per amu) over a given range and the result is plotted as a "broken line." (Used for system check out) (7) MONITOR: Provides for inspecting the peak profiles by sampling the ,$>ctrum around a given or position. The gathered dara are then typed out. (Normally used for system service or service log) (8) DISPLAY: Enables the user to display a given mass position (or N numbcrj in the center of the console oscilloscope. (Used in the adjustment of the mass spectrometer) (9) GAS: Allows the user to remot+ turn the reference gas on or off. This is helpful when operating the system from a remote position. n 31 69 :! 1'" 1212 .0600 2060 .7600 & 2507 .260I .il. 169 : 3327 1 D 1.300 # PRES Jr lo'7/orr T FACTOR 40 rlICA ' 40,&U. 123351 L/he I IONV 20 r&,;r's 691218 dafe Figure 3. A monitor plot indication of instrument scr\iccability The example of a user-computer conversation gi*:cn in Figure 2 represents the day-to-day conlput-r-r~sc:!r~her dialogue given to direct the system's olxrarion. D:?per level programming may be done at the terminal to rcdctine these functions or to add new modes. AddiIionnl b!`;tcm development may be done by the chemist-user, or his program- mer in a manner typical of general purpcse ccrnptit